Can I purchase any of the buffers that come with the MagMAX FFPE DNA/RNA Ultra Kit separately?

Protease Digestion Buffer, Binding Solution, and DNA Wash Buffer are also available as standalone Cat. No. A32796. For processing sections that are thicker than 40 µm, additional reagents would be required.

Im getting poor quality/poor purity of RNA after isolation with the RiboPure RNA Purification kit. What could be the cause of this?

- Partially degraded RNA: Use fresh tissue or cells, or use RNAlater Stabilization Solution if tissue cannot be frozen immediately.
- DNA contamination: If the sample contains organic solvents, strong buffers, or an alkaline solution, we recommend performing phase separation at 8 degrees C.
- A low A260/A280 ratio (<1.6): This indicates that an insufficient amount of the TRI Reagent was used. Incubation of the homogenate at room temperature for 5 minutes can help nucleoproteins dissociate from RNA. Phenol contamination could also cause a low absorbance ratio.

Does the DRR (DNase Binding Reagent) from RapidOut DNA Removal Kit bind to other proteins? Is the kit suitable for removal of other proteins from the reaction mixture?

We have tested the kit for DNase I removal only. Although DRR can bind to other proteins, we cannot provide any recommendations for removal of protein contaminants from the enzymatic mixtures. If the RNA sample contains a considerable amount of protein contaminants, modify the RNA purification protocol to reduce protein contamination.

I have a long double-stranded DNA fragment I would like to isolate. What product do you recommend?

For biotin-labled DNA that is less than 1 kb, we recommend you use Dynabeads M270 Streptavidin and MyOne C1 magnetic beads. We recommend our Dynabeads KilobaseBINDER Kit, which is designed to immobilize long (>1 kb) double-stranded DNA molecules. The KilobaseBINDER reagent consists of M-280 Streptavidin-coupled Dynabeads magnetic beads along with a patented immobilization activator in the binding solution to bind to long, biotinylated DNA molecules for isolation. Please see the following link (https://www.thermofisher.com/us/en/home/life-science/dna-rna-purification-analysis/napamisc/capture-of-biotinylated-targets/immobilisation-of-long-biotinylated-dna-fragments.html) for more information in regards to long biotinylated DNA fragment isolation.

Should I treat my RNA sample with DNAse for FlashTag labeling?

DNAse treatment is optional. It is not necessary for RNA samples that have trace amounts of genomic DNA contamination, but it may be beneficial for RNA samples that are highly contaminated with genomic DNA, to more accurately quantitate the RNA. After treating your RNA with DNAse, it is essential that the DNase be inactivated completely before proceeding with the FlashTag procedure, to prevent degradation of the FlashTag reagent (Vial 5). A variety of RNA purification columns/kits may be used to inactivate the DNase. Inactivation of the DNase by high temperature may not completely inactivate the enzyme.

What are different methods for cleaning up my miRNA sample?

- Precipitation of miRNA: Follow the instructions for the mirVANA miRNA Isolation Kit or the mirVANA PARIS RNA and Native Protein Purification Kit.
- Double phenol/guanidium (chaotropic reagent) extraction: Disrupt tissue with phenol/guanidine, then extract with phenol, centrifuge, remove the supernatant, and precipitate the RNA.
- Glass-fiber filter: Disrupt the tissue in a chaotropic agent, bind the RNA to a glass-fiber filter, then wash off unbound DNA/protein/salt/nucleotides and elute the RNA.

With the direct lysis approach to DNA/RNA analysis, can I use heparin- or EDTA-treated blood?

Unforutnately, the Blood-to-CT and MagMAX kits cannot be used with blood from heparin- or EDTA-stabilized tubes. The reactions require the lysis reagent found in Tempus tubes. Also, the heparin- or EDTA-stabilized tubes don't stabilize the RNA profiles, so we recommend using Tempus tubes.

I am getting shorter reads than I used to. What can cause this?

A change in the read length is usually caused by something that either changes the migration of the DNA or the efficiency of the sequencing reaction. Some of the things that can cause this are:

• Buffer: Buffer may be prepared incorrectly or on the system for too long.

• Polymer: Polymer may have been on the system the system too long, used past the expiration date, frozen/crystallized and thawed, or mixed with water or buffer during capillary fill. There may be a mismatch between polymer type selected in software and what is installed on the system.

• Environment: Instrument may be operating outside environmental specifications in the Site Preparation Guide. You may have poor/improper instrument ventilation, or air flow may be blowing directly on the system.

• DNA quality: RNA/protein contamination may be reducing the sequencing reaction efficiency. If working with PCR product, there may be carryover of primers and/or dNTPs from the PCR reaction, or the sequencing reaction cleanup is incomplete. If using a kit that has beads in it for the purification, beads may be blocking the capillary tip. If using BigDye XTerminator Purification, heating the XTerminator reagent can cause loss of smaller products.

• DNA quantity: There may be too much DNA competing for entry into the capillary with labeled product, or excessive amounts of DNA creating a temporary blockage of the capillary.

For more information on other causes of short reads and how to address each issue, please refer to the DNA Sequencing by Capillary Electrophoresis: Applied Biosystems Chemistry Guide: Second Edition (Cat. No. 4305080, Rev. C). The guide can be found by searching the Thermo Fisher Scientific website with the catalog number 4305080.